Background: Spodoptera frugiperda (Sf-9) cells are often used to produce recombinant proteins by transient expression. The culture medium contributes to the efficiency of protein production, but the effect of modern insect cell culture media on Sf-9 cells has not been evaluated for different transient expression systems. Accordingly, we compared the baculovirus expression vector system (BEVS) and lipopolyfection (LP) for the production of schistosomal ALDH and the antimicrobial peptide BR033, respectively. We used Sf-9 cells adapted to four commercially available media: TriEx ICM, ExpiSf CDM, Sf-900 II SFM and IS Sf Insect ACF. Results: During cell maintenance, the highest viable cell density was achieved using IS Sf Insect ACF, but the shortest doubling time was observed in TriEx ICM. All four media supported transient expression with standard protocols for LP and BEVS. We confirmed the typical switch in metabolic rates (glucose, lactate and amino acids) after transfection or infection, comparable to previous reports. The maximum protein production rates were 0.26 +/- 0.04 amol/(cell h) for LP and 4.63 +/- 1.85 amol/(cell h) for BEVS, both achieved using Sf-900 II SFM. Conclusions: The experiments demonstrate that the most suitable medium for transient production in Sf9 cells depends on the experimental objectives or production framework. We therefore recommend implementing the screening of different media during process development.